PROCOMCURE Biotech offers a broad selection of cell-based and biochemical screening services for the analysis of your compounds, as characterization of mode of action and functionality are crucial for the development of a viable drug.
The cell-based methods allow you to generate a cellular profile of your compound using endpoints such as NADH-, ATP-, and cAMP-content and protease activity.
The read-out is either based on a luminescent or a fluorescent reaction. We established a flexible portfolio of methods, which can be customized to your need (see also pathway reporter screening).
|Application||Marker||Detection||How it works||Notes|
|cell viability, cell toxicity, proliferation||ATP||luminescence||The amount of ATP is measured by an ATP-dependent luciferase reaction, where luciferin is metabolized and results in emission of light.||available for eukaryotic and prokaryotic cells|
|cell viability, cell toxicity, proliferation||NADH||fluorescence||Metabolically active cells are able to convert an indicator dye (by a reduction reaction), which can be detected by fluorometric measurement.||flexible incubation times possible|
|cell viability, cell toxicity||intracellular protease||fluorescence||A protease acts as marker for living cells and converts a membrane-permeable peptide into a fluorescent product and hence generates a signal which is proportional to cell viability.||can be used for normalization (well to well and plate to plate differences)|
|cell toxicity, membrane damage||DNA||fluorescence||DNA is released from dead cells and bound by the dye, which is non-toxic and non-membranepermeable. The fluorescent signal increases proportional with the released DNA, as equivalent to the number of dead cells.||can be used in addition to ATP cell viability assay|
|cell toxicity, membrane damage||extracellular protease||luminescence||Because dead cells lose their membrane integrity, a specific protease is released into the medium. This enzyme cleaves a peptide-substrate, which can then serve as substrate in a luciferase reaction. The emissioned light is a measure of the number of damaged cells.||cells stay intact, normalization possible|
|apoptosis||caspase-3 and -7||luminescence||The activity of the effector caspases -3 and -7 is a value which shows the number of apoptotic cells as a result of compound treating. The caspases cleave a peptide that can then serve as substrate in a luciferase reaction.|
|GPCR signaling pathway||cAMP content||luminescence||When a compound interacts with G-protein coupled receptors (GPCRs) linked with adenylate cyclase, the intracellular level of cAMP is altered. The cAMP acts as a stimulant for the protein kinase A (PKA) in the reagent. The active kinase metabolizes ATP that decreases the simultaneous luciferase reaction and the emitted light. That means, the lower the luminescence counts, the higher the cAMP level.|
|chymotrypsin-, trypsin- and caspase-like activity of the proteasome||substrate conversion||luminescence||All three activities of the proteasome can be measured combined in a single experiment or each of them separately. The substrate is added and processed by the proteasome to serve as reagent for a luciferase reaction.||Proteasome inhibitors are of great interest in the development of new cancer therapies.|
|cell viability, cell toxicity||MTT||colorimetric||MTT substrate is taken up by treated cells and - depending on mitochondrial integrity / activity as overall measure of cell viability - reduced to an impermeable (intracellularly accumulating) purple formazan product. Lysis of cells causes a liberation of the substrate which can be quantified via colorimetric procedures.|
|cardio toxicity||hERG channel protein||fluorescence polarization||A high-affinity fluorescent ligand (a so-called tracer) is herein displaced from the hERG channel protein (included in a membrane fraction) by compounds that bind to it, as has been validated with a panel of known blockers.||Results demonstrate a high correlation with those obtained from patch clamp techniques.|